HEPNet: A Knowledge Base Model of Human Energy Pool Network for Predicting the Energy Availability Status of an Individual

HEPNet is an electronic representation of metabolic reactions occurring within human cellular organization focusing on inflow and outflow of the energy currency ATP, GTP and other energy associated moieties. The backbone of HEPNet consists of primary bio-molecules such as carbohydrates, proteins and fats which ultimately constitute the chief source for the synthesis and obliteration of energy currencies in a cell. A series of biochemical pathways and reactions constituting the catabolism and anabolism of various metabolites are portrayed through cellular compartmentalization. The depicted pathways function synchronously toward an overarching goal of producing ATP and other energy associated moieties to bring into play a variety of cellular functions. HEPNet is manually curated with raw data from experiments and is also connected to KEGG and Reactome databases. This model has been validated by simulating it with physiological states like fasting, starvation, exercise and disease conditions like glycaemia, uremia and dihydrolipoamide dehydrogenase deficiency (DLDD). The results clearly indicate that ATP is the master regulator under different metabolic conditions and physiological states. The results also highlight that energy currencies play a minor role. However, the moiety creatine phosphate has a unique character, since it is a ready-made source of phosphoryl groups for the rapid synthesis of ATP from ADP. HEPNet provides a framework for further expanding the network diverse age groups of both the sexes, followed by the understanding of energetics in more complex metabolic pathways that are related to human disorders.     

Functional Significance of the Mitochondrial Membrane Potential

The electrical polarization of the inner mitochondrial membrane largely determines the electrochemical potential of hydrogen ifons, being thereby a significant factor in the energy transformation during oxidation of respiratory substrates and its accumulation in the form of newly synthesized ATP. However, the gradient of the electric potential on the inner mitochondrial membrane (ΔΨm) performs a number of functions not related to energy production. Even under hypoxic conditions, precluding the formation of ATP in mitochondria through oxidative phosphorylation, mitochondria maintain their ΔΨm at the expense of the hydrolysis of cellular ATP, which indicates the exceptional importance of ΔΨm for non-energetic functions of mitochondria. Among these functions, the mitochondrial inward transport of metal cations and proteins carrying a positively charged amino acid sequence and export of anions including nucleic acids possibly providing retrograde signaling, seem very important and essential for maintaining mitochondrial structure and metabolism. ΔΨm is a powerful regulator of mitochondrial generation of reactive oxygen species that perform physiological and pathological functions. And finally, ΔΨm is a critical element in the mechanism of disposal of dysfunctional mitochondria, the so-called quality control machinery of mitochondria. The disturbance of this mechanism leads to increase of heterogeneity in the population of mitochondria in the cell, and the degree of heterogeneity can be considered as an indicator of the pathological cellular phenotype. Correlation between Ψm and cell functions is difficult to identify without adequate quantitative estimates of the magnitude of ΔΨm, which are complicated due to several cellular and mitochondrial processes that affect the experimentally obtained values. Recommendations for assessing the contribution of these processes and avoiding artifacts in the measurements of ΔΨm by standard methods are given.     

KATP channel: relation with cell metabolism and role in the cardiovascular system

ATP-sensitive potassium channel (K(ATP)) is one kind of inwardly rectifying channel composed of two kinds of subunits: the pore forming subunits and the regulatory subunits. K(ATP) channels exist in the sarcolemmal, mitochondrial and nuclear membranes of various tissues. Cell metabolism regulates K(ATP) gene expression and metabolism products regulate the channel by direct interactions, while K(ATP) controls membrane potentials and regulate cell activities including energy metabolism, apoptosis and gene expression. K(ATP) channels from different cell organelles are linked by some signal molecules and they can respond to common stimulation in a coordinate way. In the cardiovascular system K(ATP) has important functions. The most prominent is that opening of this channel can protect cardiac myocytes against ischemic injuries. The sarcolemmal K(ATP) may provide a basic protection against ischemia by energy sparing, while both the sarcolemmal K(ATP) and mitochondrial K(ATP) channels are necessary for the ischemia preconditioning. K(ATP) channels also have important functions including homeostasis maintenance and vascular tone regulation under physiological conditions. Further elucidation of the role of K(ATP) in the cardiovascular system will help us to regulate cell metabolism or prevent damage caused by abnormal channel functions.     

AMPK inhibition in health and disease

All living organisms depend on dynamic mechanisms that repeatedly reassess the status of amassed energy, in order to adapt energy supply to demand. The AMP-activated protein kinase (AMPK) αβγ heterotrimer has emerged as an important integrator of signals managing energy balance. Control of AMPK activity involves allosteric AMP and ATP regulation, auto-inhibitory features and phosphorylation of its catalytic (α) and regulatory (β and γ) subunits. AMPK has a prominent role not only as a peripheral sensor but also in the central nervous system as a multifunctional metabolic regulator. AMPK represents an ideal second messenger for reporting cellular energy state. For this reason, activated AMPK acts as a protective response to energy stress in numerous systems. However, AMPK inhibition also actively participates in the control of whole body energy homeostasis. In this review, we discuss recent findings that support the role and function of AMPK inhibition under physiological and pathological states.     

Metabolic regulation ofin vivo myocardial contractile function: multiparameter analysis

To gain insite into the mechanisms of myocardial regulation as it relates to the interaction of mechanical and metabolic function and perfusion, intact animal models were instrumented for routine physiological measurements of mechanical function and for measurements of metabolism (³¹P NMR, NADH fluorescence (redox state)) and perfusion (²H NMR and Laser doppler techniques). These techniques were applied to canine and cat models of volume and/or pressure loading, hypoxia, ischemia and cardiomyopathic states. Data generated using these techniques indicate that myocardial bioenergetic function is quite stable under most loading conditions as long as the heart is not ischemic. In addition, these data indicate that there is no universal regulator and that different biochemical regulators appear to mediate stable function under different physiological and pathophysiological conditions: for example; during hypoxia, NADH redox state appears to play a regulatory role; and in pressure loading, ADP, phosphorylation potential and free energy of ATP hydrolysis as well as NADH redox state appear to be regulatory.     

Proteomics analysis of hypothalamic response to energy restriction in dairy cows

The hypothalamus is the central regulatory unit that balances a number of body functions including metabolic rate, hunger, and satiety signals. Hypothalamic neurons monitor and respond to alterations of circulating nutrients and hormones that reflect the peripheral energy status. These extracellular signals are integrated within the cell at the ATP:AMP ratio and at the level of ROS, triggering gene expression associated with glucose and lipid metabolism. In order to identify new molecular factors potentially associated with the control of energy homeostasis, metabolic adaptation, and regulation of feed intake, hypothalami from ad libitum fed and energy restricted cows were characterized using 2-DE and MALDI-TOF-MS. Among 189 different protein spots identified, nine proteins were found to be differentially expressed between groups. Beside the 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase, stress-induced phosphoprotein-1, heat shock protein 70 kDa-protein-5, dihydropyrimidinase-related protein-2, [Cu-Zn]-superoxide dismutase, ubiquitin carboxy-terminal hydrolase-L1, and inorganic pyrophosphatase were found to be up-regulated, whereas glyceraldehyde 3-phosphate dehydrogenase and aconitase-2 were down-regulated in the restricted group. In conclusion, differentially expressed proteins are related to energy and nucleotide metabolism and cellular stress under conditions of dietary energy deficiency. These proteins may be new candidate molecules that are potentially involved in signaling for maintaining energy homeostasis.     

Effects of hypo-osmotic stress on ATP release in isolated turbot (Scophthalmus maximus) hepatocytes

BACKGROUND INFORMATION: ATP is released from many cell types exposed to hypo-osmotic shock and is involved in RVD (regulatory volume decrease). Purinergic signalling events have been extensively investigated in mammals, but not in marine teleosteans. RESULTS: The effect of hypo-osmotic shock on ATP release was examined in isolated hepatocytes from turbot (Scophthalmus maximus), a marine flatfish. Hypo-osmotic stress (240 mOsm x kg(-1)) induced a significant increase in ATP efflux, and was inhibited by a potential CFTR (cystic fibrosis transmembrane conductance regulator) inhibitor, glibenclamide, but not by the MDR1 (multidrug resistance 1) P-glycoprotein inhibitor, verapamil. ATP efflux could be a cAMP-dependent process, as IBMX (isobutylmethylxanthine) and forskolin triggered the process under iso-osmotic conditions. Protein kinases, including protein kinase C, could also be involved, as staurosporine and chelerythrine inhibited the mechanism. Calcium could contribute to ATP efflux as ionomycin, a calcium ionophore, elicited a rapid release under iso-osmotic conditions, and chelation using EGTA abolished ATP release under hypo-osmotic conditions. RVD was partially abolished by apyrase, an ATP scavenger, and suramin, a purinoceptor antagonist. Moreover, hypo-osmotic shock induced a rise in intracellular calcium which could be involved in RVD. Since extracellular ATP triggered an increase in cellular free-calcium content under iso-osmotic conditions, our results could indicate that hypo-osmotic-induced ATP efflux contributes to RVD in turbot hepatocytes by stimulating purinergic receptors, which may lead to activation of a calcium signalling pathway. CONCLUSIONS: These data provide the first evidence of volume-sensitive ATP signalling for volume maintenance in a marine teleost fish cell type.     

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-ε improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-α and PKC-ε are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-ε mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.Here, we present methods for assessing the role of PKC-ε in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-ε is activated by overexpressing the constitutively active PKC-ε mutant. PKC-ε is inhibited by overexpressing the inactive mutant of PKC-ε. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F₀F₁-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F₀F₁-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.     

Regulation of neuronal bioenergy homeostasis by glutamate

Bioenergy homeostasis is crucial in maintaining normal cell function and survival and it is thus important to understand cellular mechanisms underlying its regulation. Neurons use a large amount of ATP to maintain membrane potential and synaptic communication, making the brain the most energy consuming organ in the body. Glutamate mediates a large majority of synaptic transmission which is responsible for the expression of neural plasticity and higher brain functions. Most of the energy cost is attributable to the glutamatergic system; under pathological conditions such as stroke and brain ischemia, neural energy depletion is accompanied by a massive release of glutamate. However, the specific cellular processes implicated in glutamate-dependent bioenergy dynamics are not well understood. We find that glutamate induces a rapid and dramatic reduction of ATP levels in neurons, through reduced ATP genesis and elevated consumption. ATP reduction depends on NMDA receptor activity, but is not a result of neuronal firing, gap junction-mediated leaking or intracellular signaling. Similar changes in ATP levels are also induced by synaptic glutamate accumulation following suppression of glutamate transporter activity. Furthermore, the glutamate-induced ATP down-regulation is blocked by the sodium pump inhibitor ouabain, suggesting the sodium pump as the primary energy consumer during glutamate stimulation. These data suggest the important role of glutamate in the control of cellular ATP homeostasis.     

K(ATP) channels process nucleotide signals in muscle thermogenic response

Uniquely gated by intracellular adenine nucleotides, sarcolemmal ATP-sensitive K(+) (K(ATP)) channels have been typically assigned to protective cellular responses under severe energy insults. More recently, K(ATP) channels have been instituted in the continuous control of muscle energy expenditure under non-stressed, physiological states. These advances raised the question of how K(ATP) channels can process trends in cellular energetics within a milieu where each metabolic system is set to buffer nucleotide pools. Unveiling the mechanistic basis of the K(ATP) channel-driven thermogenic response in muscles thus invites the concepts of intracellular compartmentalization of energy and proteins, along with nucleotide signaling over diffusion barriers. Furthermore, it requires gaining insight into the properties of reversibility of intrinsic ATPase activity associated with K(ATP) channel complexes. Notwithstanding the operational paradigm, the homeostatic role of sarcolemmal K(ATP) channels can be now broadened to a wider range of environmental cues affecting metabolic well-being. In this way, under conditions of energy deficit such as ischemic insult or adrenergic stress, the operation of K(ATP) channel complexes would result in protective energy saving, safeguarding muscle performance and integrity. Under energy surplus, downregulation of K(ATP) channel function may find potential implications in conditions of energy imbalance linked to obesity, cold intolerance and associated metabolic disorders.     

Assaying ATP synthesis in cultured cells: A valuable tool for the diagnosis of patients with mitochondrial disorders

Mitochondrial ATP synthase plays a central role in cell function by synthesising most of the ATP in human tissues. In different cells, active regulation of mitochondrial ATP synthase in response to cellular energy demand has been demonstrated, as well as its alteration under several pathological conditions affecting oxidative phosphorylation (OXPHOS). Traditionally, detection of OXPHOS defects is based on the spectrophotometric measurement of respiratory chain complex activities in muscle biopsies. Considering the broad clinical spectrum of mitochondrial disorders, and the difficulty in arriving at a single diagnostic method, in this study we propose measurement of ATP synthesis in mitochondria from skin fibroblasts as an effective screening tool. In the light of our results this assessment emerges as a useful marker of impaired energy production in primary OXPHOS disorders of childhood and as a tool with the potential to drive further molecular genetic studies.     

Activation of the DExD/H-box protein Dbp5 by the nuclear-pore protein Gle1 and its coactivator InsP6 is required for mRNA export

The DExD/H-box ATPase Dbp5 is essential for nuclear mRNA export, although its precise role in this process remains poorly understood. Here, we identify the nuclear pore protein Gle1 as a cellular activator of Dbp5. Dbp5 alone is unable to stably bind RNA or effectively hydrolyse ATP under physiological conditions, but addition of Gle1 dramatically stimulates these activities. A gle1 point mutant deficient for Dbp5 stimulation in vitro displays an mRNA export defect in vivo, indicating that activation of Dbp5 is an essential function of Gle1. Interestingly, Gle1 binds directly to inositol hexakisphosphate (InsP6) and InsP6 potentiates the Gle1-mediated stimulation of Dbp5. Dominant mutations in DBP5 and GLE1 that rescue mRNA export phenotypes associated with the lack of InsP6 mimic the InsP6 effects in vitro. Our results define specific functions for Gle1 and InsP6 in mRNA export and suggest that local activation of Dbp5 at the nuclear pore is critical for mRNA export.     

Physiological and pathophysiological roles of ATP-sensitive K+ channels

ATP-sensitive potassium (K(ATP)) channels are present in many tissues, including pancreatic islet cells, heart, skeletal muscle, vascular smooth muscle, and brain, in which they couple the cell metabolic state to its membrane potential, playing a crucial role in various cellular functions. The K(ATP) channel is a hetero-octamer comprising two subunits: the pore-forming subunit Kir6.x (Kir6.1 or Kir6.2) and the regulatory subunit sulfonylurea receptor SUR (SUR1 or SUR2). Kir6.x belongs to the inward rectifier K(+) channel family; SUR belongs to the ATP-binding cassette protein superfamily. Heterologous expression of differing combinations of Kir6.1 or Kir6.2 and SUR1 or SUR2 variant (SUR2A or SUR2B) reconstitute different types of K(ATP) channels with distinct electrophysiological properties and nucleotide and pharmacological sensitivities corresponding to the various K(ATP) channels in native tissues. The physiological and pathophysiological roles of K(ATP) channels have been studied primarily using K(ATP) channel blockers and K(+) channel openers, but there is no direct evidence on the role of the K(ATP) channels in many important cellular responses. In addition to the analyses of naturally occurring mutations of the genes in humans, determination of the phenotypes of mice generated by genetic manipulation has been successful in clarifying the function of various gene products. Recently, various genetically engineered mice, including mice lacking K(ATP) channels (knockout mice) and mice expressing various mutant K(ATP) channels (transgenic mice), have been generated. In this review, we focus on the physiological and pathophysiological roles of K(ATP) channels learned from genetic manipulation of mice and naturally occurring mutations in humans.     

Redox modulation of cellular signaling and metabolism through reversible oxidation of methionine sensors in calcium regulatory proteins

Adaptive responses associated with environmental stressors are critical to cell survival. Under conditions when cellular redox and antioxidant defenses are overwhelmed, the selective oxidation of critical methionines within selected protein sensors functions to down-regulate energy metabolism and the further generation of reactive oxygen species (ROS). Mechanistically, these functional changes within protein sensors take advantage of the helix-breaking character of methionine sulfoxide. The sensitivity of several calcium regulatory proteins to oxidative modification provides cellular sensors that link oxidative stress to cellular response and recovery. Calmodulin (CaM) is one such critical calcium regulatory protein, which is functionally sensitive to methionine oxidation. Helix destabilization resulting from the oxidation of either Met(144) or Met(145) results in the nonproductive association between CaM and target proteins. The ability of oxidized CaM to stabilize its target proteins in an inhibited state with an affinity similar to that of native (unoxidized) CaM permits this central regulatory protein to function as a cellular rheostat that down-regulates energy metabolism in response to oxidative stress. Likewise, oxidation of a methionine within a critical switch region of the regulatory protein phospholamban is expected to destabilize the phosphorylation-dependent helix formation necessary for the release of enzyme inhibition, resulting in a down-regulation of the Ca-ATPase in response to beta-adrenergic signaling in the heart. We suggest that under acute conditions, such as inflammation or ischemia, these types of mechanisms ensure minimal nonspecific cellular damage, allowing for rapid restoration of cellular function through repair of oxidized methionines by methionine sulfoxide reductases and degradation pathways after restoration of normal cellular redox conditions.     

Differential effects of bcl-2 on cell death triggered under ATP-depleting conditions

The intracellular ATP concentration decides on the onset of either apoptosis or necrosis in Jurkat cells exposed to death stimuli. Bcl-2 can block apoptotic demise, which occurs preferably under conditions of high cellular ATP levels. Here, we investigated the effects of Bcl-2 on the necrotic type of cell demise that prevails under conditions of energy loss. ATP levels were modulated by using mitochondrial inhibitors, such as rotenone or S-nitrosoglutathione, in medium either lacking glucose or supplemented with glucose to stimulate glycolytic ATP generation. Under conditions of ATP depletion, staurosporine (STS) induced >90% necrosis in vector control-transfected cells, whereas bcl-2-transfected cells were protected. Thus, the antiapoptotic protein Bcl-2 can reduce the overall amount of cell death in ATP-depleted cells regardless whether it occurs by apoptosis or necrosis. Cytochrome c release, normally preceding STS-induced necrosis, was also inhibited by Bcl-2. However, Bcl-2 did not prevent an initial STS-induced drop of the mitochondrial membrane potential (DeltaPsi(m)). Therefore, the mechanisms whereby Bcl-2 prevents cell death and favors retention of cytochrome c in the mitochondria require neither the maintenance of mitochondrial DeltaPsi nor the maintenance of normal ATP levels.     

Energy Metabolism Response to Low-Temperature and Frozen Conditions in Psychrobacter cryohalolentis

Studies of cold-active enzymes have provided basic information on the molecular and biochemical properties of psychrophiles; however, the physiological strategies that compensate for low-temperature metabolism remain poorly understood. We investigated the cellular pools of ATP and ADP in Psychrobacter cryohalolentis K5 incubated at eight temperatures between 22°C and −80°C. Cellular ATP and ADP concentrations increased with decreasing temperature, and the most significant increases were observed in cells that were incubated as frozen suspensions (<−5°C). Respiratory uncoupling significantly decreased this temperature-dependent response, indicating that the proton motive force was required for energy adaptation to frozen conditions. Since ATP and ADP are key substrates in metabolic and energy conservation reactions, increasing their concentrations may provide a strategy for offsetting the kinetic temperature effect, thereby maintaining reaction rates at low temperature. The adenylate levels increased significantly <1 h after freezing and also when the cells were osmotically shocked to simulate the elevated solute concentrations encountered in the liquid fraction of the ice. Together, these data demonstrate that a substantial change in cellular energy metabolism is required for the cell to adapt to the low temperature and water activity conditions encountered during freezing. This physiological response may represent a critical biochemical compensation mechanism at low temperature, have relevance to cellular survival during freezing, and be important for the persistence of microorganisms in icy environments.     

Inhibition of an iron-responsive element/iron regulatory protein-1 complex by ATP binding and hydrolysis

Iron regulatory protein-1 binding to the iron-responsive element of mRNA is sensitive to iron, oxidative stress, NO, and hypoxia. Each of these agents changes the level of intracellular ATP, suggesting a link between iron levels and cellular energy metabolism. Furthermore, restoration of iron regulatory protein-1 aconitase activity after NO removal has been shown to require mitochondrial ATP. We demonstrate here that the iron-responsive element-binding activity of iron regulatory protein is ATP-dependent in HepG2 cells. Iron cannot decrease iron regulatory protein binding activity in cell extracts if they are simultaneously treated with an uncoupler of oxidative phosphorylation. Physiologic concentrations of ATP inhibit iron-responsive element/iron regulatory protein binding in cell extracts and binding of iron-responsive element to recombinant iron regulatory protein-1. ADP has the same effect, in contrast to the nonhydrolyzable analog adenosine 5'-(beta,gamma-imido)triphosphate, indicating that in order to inhibit iron regulatory protein-1 binding activity, ATP must be hydrolyzed. Indeed, recombinant iron regulatory protein-1 binds ATP with a Kd of 86+/-17 microM in a filter-binding assay, and can be photo-crosslinked to azido-ATP. Upon binding, ATP is hydrolyzed. The kinetic parameters [Km=5.3 microM, Vmax=3.4 nmol.min(-1).(mg protein)(-1)] are consistent with those of a number of other ATP-hydrolyzing proteins, including the RNA-binding helicases. Although the iron-responsive element does not itself hydrolyze ATP, its presence enhances iron regulatory protein-1's ATPase activity, and ATP hydrolysis results in loss of the complex in gel shift assays.     

Physiological roles of the mitochondrial permeability transition pore

Neurons experience high metabolic demand during such processes as synaptic vesicle recycling, membrane potential maintenance and Ca²⁺ exchange/extrusion. The energy needs of these events are met in large part by mitochondrial production of ATP through the process of oxidative phosphorylation. The job of ATP production by the mitochondria is performed by the F₁F_O ATP synthase, a multi-protein enzyme that contains a membrane-inserted portion, an extra-membranous enzymatic portion and an extensive regulatory complex. Although required for ATP production by mitochondria, recent findings have confirmed that the membrane-confined portion of the c-subunit of the ATP synthase also houses a large conductance uncoupling channel, the mitochondrial permeability transition pore (mPTP), the persistent opening of which produces osmotic dysregulation of the inner mitochondrial membrane, uncoupling of oxidative phosphorylation and cell death. Recent advances in understanding the molecular components of mPTP and its regulatory mechanisms have determined that decreased uncoupling occurs in states of enhanced mitochondrial efficiency; relative closure of mPTP therefore contributes to cellular functions as diverse as cardiac development and synaptic efficacy.